Human bones are always remodelling by the repeated process of resorption and reconstitution. In the process, osteoblasts and osteoclasts are considered to be the calls mainly in charge of bone formation and bone resorption, respectively. A typical example of disease caused by abnormal bone metabolism proceeded by the bone cells is osteoporosis. The disease is known to be provoked by the condition in which bone resorption by osteoclasts exceeds bone formation by osteoblasts, but the mechanism of osteoporosis has not yet been completely elucidated. Osteoporosis causes pain in the bone and makes the bone fragile, leading to fracture. Since osteoporosis increases the number of bedridden old people, it has become a social issue with the increasing number of old people. Therefore, efficacious drugs for the treatment of the disease are expected to be developed. Bone mass reduction caused by the abnormal bone metabolism is thought to be treated by inhibiting bone resorption, improving bone formation, or improving the balanced metabolism.
Bone formation is expected to be promoted by stimulating growth, differentiation, or activation of osteoblasts. Recently, cytokines which stimulates growth or differentiation of osteoblasts have been attracted public attention and have been intensively studied. Many cytokines are reported to stimulate the growth of osteoblasts, i.e. fibroblast growth factor (FGF) (Rodan S. B. at al., Endocrinology vol.121, p1917, 1987), insulin-like growth f actor-I (IGF-I) (Hock J. M. et al., Endocrinology vol. 122, p254, 1988), insulin-like growth factor-II (IGF-II) (McCarthy T. et al., Endocrinology vol.124, p301, 1989), and bone morphogenetic protein (BMP) (Sampath T.K. etl al., J. Biol Chem. vol.267, p20532, 1992, Knutsen R. et al., Biochem. Biophys. Res. Commun. vol.194, p1352, 1993, and Akira Yamaguchi et al., Zikken Igaku vol.10, p2003, 1992). Many cytokines are also reported to stimulate the differentiation of osteoblasts, i.e. transforming growth factor-s (TGF-P) (Centrella M. et al., J. Biol. Chem. vol.262, p2869, 1987), insulin-like growth factor (IGF), and bone morphogenetic protein (Takuwa :Y. et al., Biochem. Biophys. Res. Commun. vol.174, p96, 1991, and Knutsen R. et al., Biochem. Biophys. Res. Commun. vol.194, p1352, 1993). These cytokines are expected to be efficacious drugs for improving bone mass by stimulating bone formation; some of the cytokines such as bone morphogenetic proteins are now investigated in clinical trials for their effects to cure the patients with bone diseases.
Examples of drug products now clinically utilized for the treatment of bone diseases and for shortening the treatment period are dihydroxy vitamine D.sub.3, calcitonin and its derivatives, hormones such as estradiol, lprif lavon, and calcium preparations. However, these drug products do not provide satisfactory therapeutic effects, and novel drug substances have been expected to be developed. As mentioned, bone metabolism is controlled in the balance between bone resorption and bone formation. Therefore, cytokines which stimulate osteoblast growth and osteogenesis are expected to be developed as drug for the treatment of bone diseases such as osteoporosis.
Disclosure of the Invention
The inventors have intensively searched for osteoblast growth factors, and have found a novel osteoblast growth factor. The inventors have also established methods for accumulating the protein in a high concentration and purifying it efficiently.
A cDNA clone encoding this protein was isolated by using the partial amino-acid sequences of the native bOGF-II protein. Moreover, bOGF-II was produced by the genetic engineering techniques with this cDNA. The object of the invention is to provide a novel osteoblast growth factor.(protein) and methods for efficiently producing the protein.
The inventors screened animal cells conditioned media, and have found the osteoblast growth factor in human fibroblast IMR-90 (ATCC-CCL186) conditioned medium. The inventors examined the culture conditions of IMR-90 cells, and have established the method for culturing the cells on alumina ceramic fragments to accumulate the osteoblast growth factor in a high concentration in the culture medium. The inventors found the method to purify bOGF-Il efficiently by a combination of ion-exchange column and/or heparin column.
Moreover, the inventors determined the amino acid sequences of the bOGF-II protein, designed the primers based on these amino acid sequences, and obtained a cDNA fragments of bOGF-II from a cDNA library of IMR-90 cells.
A cDNA clone encoding the full length protein of the current invention was isolated from a cDNA library of IMR-90 cells by hybridization using the cDNA fragment as a probe. Furthermore, the inventors established a method for producing recombinant bOGF-II in the culture media of the cells which was transformed by expression vector containing the cDNA.
This invention relates to a protein characterized by the following features: (1) derived from human fibroblast cells, (2) molecular weight of ca. 15kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions, (3) a high affinity for cation exchanger and heparin, (4) decrease in osteoblast growth activity by heating at 70.degree. C. for 10 minutes, and (5) inactivation by heating at 90.degree. C. for 10 minutes. bOGF-II in the present invention is apparently different from known osteoblast growth factors in N-terminal amino acid sequence. The N-terminal amino acid sequences of bOGF-II are shown in Sequence Number 1 and 2.
The invention also relates to a method for producing bOGF-II, comprising: (1) culturing human fibroblasts, (2) treating the culture medium through a heparin-column, (3) eluting an adsorbent fraction, (4) treating the eluate through an anion-exchange column to obtain a non-adsorbent fraction, (5) applying the fraction to a cation-exchange column, and (6) purifying the objective protein through a heparin-column.
Purification procedure according to the invention includes any means having the same effect as that obtained by the method for mixing a culture medium with heparin-Sepharose, etc. in a batchwise operation and for treating through a column, as well as a simple method for flowing a culture medium through heparinSepharose column, etc.
bOGF-II can be efficiently isolated from a culture medium of human fibroblasts at a high yield according to the invention. Isolation of bOGF-II is based on general means for purifying proteins from biomaterials, utilizing physical and chemical properties of the objective protein bOGF-II. For example, concentration procedure includes general biochemical technique such as ultrafiltration, lyophilization, and dialysis. Purification procedure includes combinations of several technique for purifying proteins, such as ion-exchange chromatography, affinity chromatography, gel filtration chromatography, hydrophobic chromatography, reversed-phase chromatography, and preparative gel electrophoresis. Human fibroblast is preferably IMR-90. The culture medium of human fibroblast cell IMR-90 is obtained by absorbing human fibroblast cell IMR-90 on ceramic, and culturing in DMEM medium supplemented with 5% fetal calf serum in a roller bottle in stationary state for about a week. For purification, 0.1% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate) is preferably added to a buffer as surfactant.
The protein of the invention is purified by applying the culture medium to a heparin-column (heparin-Sepharose CL6B, manufactured by Pharmacia), eluting with 10 mM Tris-HCl buffer containing 2 M NaCl, pH 7.5 applying the diluted fraction to anion-exchange column (Hiload-Q/FF, manufactured by Pharmacia), collecting an non-adsorbent fraction, and applying the obtained fraction to S cation-exchange column (Hiload-S/HP, manufactured by Pharmacia) to fractionate three peaks, bOGF-I (0.15 M NaCl), bOGF-II (0.35 M NaCl), and -bOGF-III (0.55 M NaCl) in the order that the activity is eluted in lower concentrations of NaCl. bOGF-II can be isolated by the following repeated heparin column chromatography (heparin-5PW, manufactured by Toso Co.), and can be identified by the previously described properties. It is likely that the protein bOGF-III is basic fibroblast growth factor from the results that it is inactivated by heating at 70.degree. C. for 10 minutes, it is eluted by ca. 1.8 M NaCl from heparin 5PW column, and it is inactivated by anti-basic fibroblast growth factor antibody.
Furthermore, a method for producing recombinant bOGF-II was established. The method includes the following three steps; First, amino-acid sequences of bOGF-II are used to design oligonucleotide primers. Second, a bOGF-II cDNA fragment is obtained by PCR amplification using the primers. Finally, the cDNA clone encoding the full length bOGF-II is isolated from a cDNA library of IMR-90 cells by hybridization using the cDNA fragment as a probe. Moreover, bOGF-II is recovered and purified from culture medium or the cells by culturing host cells selected from eukaryote such as mammalian cells (e.g. chinese hamster ovary cell) or prokaryote such as bacteria (e.g. E. coli.), which are transfected by vector, having expression promoter and the cDNA coded full-length bOGF-II.
The invention relates to proteins having an activity to stimulate growth of osteoblasts, containing the described amino acid sequence as a part, or having a homology with the described amino acid sequence more than 801, and cDNA of the protein.
OGF activity can be evaluated by utilizing osteoblastic cell lines or normal asteoblasts as target cells and by measuring an increased incorporation .sup.3 H-thymidine to the cells. The target cell is preferably mouse osteoblastic cell line MC3T3-E1 (J. Oral. Bio. Cell. Biol. 96, 191, 1983). The cell is reported to be responsive to vitamin D.sub.3 and parathyroid hormone and to grow up to be calcificated in vitro in a manner similar .sub.to that in vivo. The OGF activity is preferably measured with a serum-free medium, and can be exactly evaluated at high sensitivity by measuring incorporation of .sup.3 H-thymidine.
bOGF-II is useful as a pharmaceutical composition for treating or improving decreased bone mass such as in osteoporosis and other diseases with abnormal bone metabolism, or as antigen for immunological diagnosis of the diseases.
bOGF-II is formulated to be pharmaceutical preparation, and can be orally or parenterally administered. The preparation comprises bOGF-II as a an effective ingredient, and is safely administered to human beings. Examples of the pharmaceutical preparation include compositions for injection or intravenous drip, suppositories, nasal preparations, sublingual preparations, and tapes for percutaneous absorption. The preparation for injection is a mixture of bOGF-II in pharmacological effective amount and pharmaceutically-acceptable carrier. The carrier is vehicle/activator which is generally added to compositions for injection, e.g. amino acids, saccharides, cellulose derivatives, and other organic/inorganic compounds. When mixing bOGF-II with the vehicle/activator to prepare injections, pH adjustor, buffer, stabilizer, solubilizing agent, etc. may be added as needed.